Barbieri J T, Moloney B K, Mende-Mueller L M
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
J Bacteriol. 1989 Aug;171(8):4362-9. doi: 10.1128/jb.171.8.4362-4369.1989.
The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.
百日咳毒素S-1亚基（rS-1）的结构基因和S-1亚基的催化C180肽（C180肽）分别独立亚克隆至大肠杆菌中tac启动子的下游。这两种构建体都包含编码S-1亚基预测前导序列的DNA，该序列插入在tac启动子和结构基因之间。含有编码rS-1和C180肽质粒的大肠杆菌产生了一种能与抗百日咳毒素抗体发生反应且分子量与克隆基因相对应的肽；观察到rS-1有一些降解。含有编码rS-1和C180肽质粒的大肠杆菌提取物具有ADP-核糖基转移酶活性。亚细胞分级分离显示rS-1和C180肽都存在于周质中，这表明大肠杆菌识别百日咳毒素肽前导序列。C180肽氨基末端的蛋白质序列与百日咳博德特氏菌产生的天然S-1亚基相同，这表明大肠杆菌前导肽酶正确加工了百日咳毒素肽前导序列。将先前显示可降低ADP-核糖基转移酶活性的第26位残基（C180I-26）和第139位残基（C180S-139）的两个单氨基酸取代引入C180肽中。C180I-26具有C180肽约1%的NAD-糖水解酶活性，这表明色氨酸26在NAD与C180肽的相互作用中起作用。相比之下，C180S-139具有与C180肽基本相同水平的NAD-糖水解酶活性，这表明谷氨酸139在NAD的相互作用中不起作用，但在ADP-核糖基转移酶反应的后续步骤中起作用。
